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APPLICATIONS OF FIBER-OPTICS AND DIODE ARRAYS FOR THE MEASUREMENT OF DYNAMIC CHANGES IN LIQUID AND SOLID THIN FILMS
No full textA method is developed to measure transmittance and reflectance pattern either to calculate
changes in concentration according to Lambert-Beer's law or to determine the product of
optical pathlength times refractive index. Both can be obtained in thin films, polymers or in
flow systems. The real as well as the imaginary part of the dispersion curve can be used.
The apparatus contains a polychromatic light source, a combined dispersive element, and a
diode array both adjusted in a ceramic body inaffected by surrounding conditions (fig. 1). An
Y-fiber optic (reflectance) or two single fibers respectively (transmittance) are used to observe
the sample, which is a newly developed ultra microflow cell or a thin film. Process control,
evaluation and graphics are obtained by a specially programmed work station, \containing a
68020/68881 on a VME-bus system, using the real time operating system PDOS. Fig. 2 gives the
optical pathway from the light source (in
the cell or the thin film) to the polychromator
of the diode array
ANREICHERUNG VON ENZYMEN AUS PFLANZEN
No full textDie Enzymtechnologie gewinnt im Bereich der Lebensmittelindustrie,
und speziell in der Zuckerindustrie, zunehmend an Bedeutung. Als
Beispiel sei hier nur die enzymatische Umwandlung von Saccharose
(zur Palatinose oder Leucrose) genannt (1, 2). Daneben scheint es
auch lohnenswert, die Gewinnung von Enzymen aus landwirtschaftlichen Rohstoffen, die in großen Mengen und zu günstigen Preisen
verfügbar sind, zu untersuchen. Insbesondere pflanzliche Enzyme,
die nicht ohne weiteres aus Mikroorganismen gewonnen werden können, dürften von Interesse sein. Allerdings muß das Problem der
Anreicherung aus niedrigen Konzentrationen gelöst werden
DETERMINATION OF PROTEIN STRUCTURES BY NMR-APPROACHES TOWARDS EXTENDING THE LIMITS
No full textNuclear magnetic resonance spectroscopy has evolved into a powerful technique for structure determination in solution. Key advances have been the introduction of two dimensional experiments, high field superconducting magnets and computational proceedures for converting the experimental data into three-dimensional structures. Present attempts to extendthe limits of the approach, both with respectto the molecular weight range of molecules that can be studied, as wellasto the precision with whichthosestructures can be obtained are discussed
NEW DEVELOPMENTSIN PROTEIN CRYSTALLOGRAPHY
No full textProtein crystallography is currently undergoing a rapid change in many different
ways. One development is the explosion of interest by molecular biologists and immunologists
since knowledge of protein sequences, obtained via DNA sequencing, is expanding
rapidly, but does often not increase immediately insight into the functioning of the
protein. Another change is the recombinant DNA technique which make it possible to
obtain large amounts of proteins which were previously only available in minute quantities.
A third change is the wide-spread awareness that detailed knowledge of wellselected
protein structures is a promising starting point for designing new pharmaceuticals
and vaccines, for obtaining new proteins via protein engineering techniques and
for inspiring synthetic chemists in their biomimetic endeavours.
At the same time many technical aspects of protein crystallography are undergoing
a rapid development. Someof them will be describedin this paper.
Crystal structures of proteins can be obtained currently in two quite different
ways: (i) the "multiple isomorphous replacement" (MIR) method [1-3] for de novo structure
determinations, often using additional anomalous scattering information (MIRAS)
[4,5]; and, (ii) the "molecular replacement" (MR) method [6-8] for solving new structures
related to a known structure. We will discuss the steps involved in obtaining high
resolution X-ray structures as outlined in Figure 1. A detailed account of these steps
can be found in two volumes of Methods of Enzymology [9]
PROTEIN ENGINEERING OF HUMAN-LYSOZYME
No full textA gene encoding human-lysozyme was chemically synthesized and expressed
both in E. coli and S. cerevisiae. The gene product expressed in E.coli formed
insoluble material and had no enzymatic activity . For the expression in S.
cerevisiae a signal sequence of chicken-lysozyme was attached.
Prehuman-lysozyme expressed in yeast was properly processed and secreted
outside the cell. Amino acid residues of catalytic and recognition sites
(Glu35, Asp53, Tyr63, Trp64, Trp109) of human-lysozyme were changed by site
specific mutagenesis and their influence to the enzymatic activity was
examined. The surface charge of the enzyme has great effects on enzymatic
activity to charged substrates. By increasing or decreasing the surface
charge of human-lysozyme the optimum ionic strength or PH was shifted
GENETIC ENGINEERING OF PROTEASE INHIBITORS Alpha1-ANTITRYPSIN AND HIRUDIN
No full textSite-directed mutagenesis was employed to express different
variants of a,antitrypsin in a recombinant strain of
Escherichia coli. The first set of variants was designed to
render the inhibitor stable under oxidative conditions which
reduce the activity of the natural molecule. This was achieved by
replacing the methionine residue in position 358 (Pl position) by
either valine or leucine. In vitro testing of the variant
inhibitors under conditions which mimic the in vivo situation of
oxidative stress in the epithelial lining fluid of the lung of
cigarette smokers confirmed that they retained their inhibitory
efficiency against neutrophil elastase.
Furthermore site-directed mutagenesis was used to replace
the methionine??® residue with an arginine in order to change
the inhibitory specificity of a,antitrypsin from neutrophil
elastase to a-thrombin. The design of this inhibitor was based
upon the known specificity of a-thrombin for arginine and also
upon the presence of an arginine residue in the Pl position of
the natural a-thrombin inhibitor antithrombin III. Subsequent in
vitro and in vivo evaluation of this variant confirmed that a
potent inhibitor of a-thrombin had been designed.
Based on further sequence homology studies with other
members of the serine protease inhibitor (serpin) family another
a@,antitrypsin variant with more pronounced inhibitory effects
on plasma kallikrein and factor XIIa was designed. This was
achieved by replacing the proline357 residue in the
arginine358 variant with an alanine. The double variant matched
the Cl-inhibitor in its P2 and P1 positions and proved to be more
effective against plasma kallikrein and factor XIIa both in vitro
and in vivo than the mutant with only an arginine?>® residue.
In a separate set of experiments variants of the naturally
occurring a-thrombin inhibitor hirudin were designed and
expressed in a recombinant yeast strain. Site-directed
mutagenesis experiments established the importance of having a
basic residue such as lysine or arginine in position 47 of the
inhibitor to obtain efficient thrombin inhibition. In addition it
was shown that a replacement of lysine?5 with a threonine
residue did not alter the inhibition efficiency thus indicating
that the surface loop region around this residue is not involved
in the interaction with a-thrombin
ENZYME SENSORS FOR PROCESS CONTROL OF CELL CULTURES
No full textEnzyme sensors for D-glucose, L-lactate and L-glutamine were tested for
monitoring animal cell cultures. By coupling with FIA-techniques an online
process control could be realize
EXTRAKTION VON METABOLITEN
No full textReaktivextraktionsverfahren zeichnen sich gegentiber der Ublichen, physikalischen
Extraktion durch die Erreichbarkeit höherer Verteilungskoeffizienten
und größerer Selektivität aus. Mit der Untersuchung der
Reaktivextraktion von Penicillin G wird die sinnvolle Anwendung dieser
Technik in der Aufarbeitung biologischer Medien demonstriert.
Neben der Extraktion von Modellmedien konnte auch die praktisch quantitative
Abtrennung von Penicillin G aus realen Fermentationsmedien
erreicht werden. Die bei dem heute industriell eingesetzten Extraktionsverfahren
auftretenden Produktverluste werden dabei vermieden
MIZELLARE UND MESOMORPHE STRUKTUREN UND IHRE QUELLBARKEIT, EIN BEITRAG ZUR SOLUBILISIERUNG UND KOAZERVIERUNG VON HYDROPHOBEM, BIOLOGISCHEM MATERIAL
No full textDie Solubilisierung hydrophoben Materials wie beispielsweise die Solubilisierung
hydrophober Proteine kann mit Hilfe von wässrigen Polyglykoletherlösungen durchgeführt
werden. Die hydrophoben Substanzen werden in den Kohlenwasserstoffbereichen
hydrophiler, lamellarer Strukturen gespeichert, die dadurch quellen und aufgeweitet
werden, Übersteigt die Aufnahme organischer Substanzen das hydrophile - hydrophobe
Gleichgewicht, bilden sich Emulsionströpfchen, die zur Koaleszenzverhinderung mit
einem die Emulsionströpfchen umhüllenden Film ausgestattet werden müssen.
Für die Trennung hydrophober Materialien von hydrophilen ist eine Mischungslücke
erforderlich, die bei Polyglykolethern leicht zu realisieren ist. In der tensidreichen
Koazervatphase nehmen hydrophobe Lamellen, die durch Wasserabspaltung aus
hydrophilen Strukturen entstanden sind, die organische Substanz auf
Title - Preface - Contents - List of Authors
No full textThe field of biosensorsis one of the manyareas in biotechnology which currently exhibit
characteristics of exponential growth. While until a few years ago still one of the
outposts of enzymology, nowadaysresearch centers around the world and many
companies (venture business as well as the “establishment”) have proclaimed their
interest in this area, and in fact recent years have seen a sharpincreasein related
publications, patents and in biosensor equipment appearing on the open market.
According to Chemical Abstracts, 332 communications in this field were published in
1985, with 7 countries contributing about 80%of the worldwide activities (and more
than 95%of the 114 patents), and Japantaking a clear lead (Fig. 1).
Amore detailed analysis revealed that — apart from a “learning curve”-type progressin
applying established principles to biosensor construction, new developments in
transducer technology (e.g. the application of FETs, fiber optics and piezocrystals),
flow injection methods, and creativity in biomolecule selection (e.g. antibodies, membrane
constituents, organelles, tissues and receptors) have resulted in a host of new
possibilities which do not yet show anysigns of becoming exhausted.
In this situation, it was decided that the Gesellschaft fiir Biotechnologische Forschung
(GBF), which is the national research institute for biotechnological research in the
Federal Republic of Germany, should start a majoreffort to enterthis field, and that an
international workshop would provide the most appropriate opportunity to meet the
leading authorities and to shape our own research program.
In a boomingfield (as biotechnologyis today), it is often quite difficult to identify the
“trendsetter laboratories” in a discipline where oneis notyet sufficiently familiar.
Fortunately, the “Biotechnology Abstract Analyzer”, a proprietary computer-based
evaluation of publishedliterature (based on “CAS Online”) which is designedto assist
in the perceptionof leaders, trends and cooperations, provided us with a goodlead in
Additional formats of this computeranalysis (which are not indicated here) allowed us
to identify the research fields emphasized by the leaders; a tentative program for the
planned workshop emerged asa result.
A “tour de force” visit to dominant laboratories (10 in the USA,7 in Japan, and 3 in
Europe, in a total of only 8 working days), then gave the opportunity for the personal
discussions vital to shape thefinal program of the workshop. Weat GBF are most obliged
to all colleagues who accepted myvisits in 1986, both for their kind patience with
a “newcomer”, and for their extremely valuable advice with respect to the workshop
program. Special thanks are due to M. Aizawa and to the co-organizers of the workshop
and co-editors of this book, G. Guilbault (New Orleans), |. Karube (Tokyo), H.-L.
Schmidt (Weihenstephan) and L. Wingard (Pittsburgh).
As a result of these preparatory steps, the workshop, which tookplace at the GBF in
Braunschweig from June 23-26, 1987, was considered by mostparticipants to have
provided a practically complete overview of the state-of-the-art in the field of biosensors.
Two features of the format of the meeting deserve special reference:
1. Following a suggestion of L. Wingard, 3 potentially relevant lectures on “longrange”
topics wereincluded(“Horizon Lectures”); they permitted timely and stimulating
breaks in an otherwise extremely busy schedule.
2. All researchers interested to join the conference but who had not beenspecifically
invited as speakers were requested (and agreed) to show postersto facilitate the
communication and exchangeof ideas. The 37 posters which included displayed
information from 16 companieslocated in 5 countries complemented the lectures
in an ideal way.
We at GBF would onceagain like to extend our thanks and appreciation to all speakers
and exhibitors of posters, and to the many people at GBF who helped so much in organizing
the event*. We hopethat the reader of the Proceedingswill find this book as
stimulating as the participants did the Workshopitself; both of which emphasizein
their own right a rapidly diversifying field which is gaining momentum in terms of new
concepts and industrial applications